Highthroughput Screening of Soluble Recombinant Proteins Protein Science

High-throughput Screening of Soluble Recombinant Proteins Protein Science, 2002 , vol 11 , 1714 -1719 YAN-PING SHIH, 1 WEN-MEI KUNG, 1 JUI-CHUAN CHEN, CHIA-HUI YEH, ANDREW H. -J. WANG Speaker: Chung-Sheng Liu 2002/10/29

Introduction v The function of a gene is manifested by the protein it encodes. v Genome sequencing of many organisms has led to the concept of analyzing protein function on a genome-wide scale. v Structural genomics and proteomics, therefore, have become major research foci.

Cloning and expression in Escherichia coli v Advantage： - has relatively simple genetics, is well characterized - has a relatively rapid growth rate - has few post-translational protein modifications v Disadvantage： - expressing heterologous proteins in E. coli are frequently expressed as insoluble aggregated folding intermediates, known as inclusion bodies

Blunt-End PCR product 5’AATTC 3’TTAAG CTCGA 3’ GAGCT 5’ Enzyme digestion 5’AATTC 3’G Subcloning C 3’ GAGCT 5’

General cloning strategy of HP p. ET Top 10 T-vector Ligation His S-tag BL 21 Target Protease cleavage site

Sticky-End PCR: New Method for Subcloning T 4 polynucleotide kinase + ATP Ligation with vectors which had been double digested and were dephosphorylated by calf intestine alkaline phosphatase ~25% final product carries two cohesive ends

Sticky-end PCR and directional cloning methods’ advantages v Simpler: It allows direct cloning of PCR products into multiple expression vectors. v It is more accurate in theory and also in practice.

Eight different fusion protein expression vectors and Three type host strains JM 109(DE 3): both plasmid preparation and protein expression BL 21 -Gold(DE 3) or -Condon. Plus(DE 3): alleviate codon bias or toxicity

Eight fusion protein expression vectors • His (histidine) : p. ET-28 a (Novagen) • Trx (thioredoxin) : p. ET-32 a (Novagen) • Nus. A (Nus. A protein) : p. ET-43. 1 a (Novagen) • CAP (cellulose-associated protein) : p. ET-35 b 2 (Novagen) • CBP (calmodulin binding protein) : p. ET-22 b+ (Novagen) • Intein (chitin binding tag) : p. TYB 11(NEB) • MBP (maltose-binding protein) : p. MAL-C 2 XC (NEB) • GST (glutathione S-transferase) : p. GEX-4 T (Pharmacia)

~40 genes into eight expression vectors ( >300 cloning reactions) >95% success cloning rate >80% highly express and soluble these target protein: 9100 k. D

Fusion Proteins Solubility Test Lane 1: whole cell lysates of induced cells Lane 2: whole cell lysates of uninduced cells Lane 3: soluble proteins with induction

Statistical Analysis of Soluble Protein Ratio Nus. A(54 k. D): 60% #90, 000 g ultracentrifugal force: MBP (42 k. D): 60% eliminate partially folded protein aggregates GST (24 k. D): 38%

Two steps of affinity purification N terminal- Tag Target His -C terminal Protease cleavage site fusion protein: 5~20 mg/l LB >90% purity

Summary v No restriction digestion of the PCR products. v All target genes can be directionally cloned into eight different fusion protein expression vectors using two universal restriction sites and with high efficiency (>95%).

Summary v 80% of the genes screened show high levels of expression of soluble products in at least one of the eight fusion protein constructs. v High-speed centrifugation in a 96 -tube format is well suited for automation and is applicable for the production of large numbers of proteins for genome-wide analysis.

Thanks for your attention